Multivariate techniques such as classical least squares (CLS), partial least squares (PLS) and principal component regression (PCR), have often been applied in the spectroscopic analysis of samples that contain multiple components of interest. These techniques provide accurate estimates of the components' concentrations when certain assumptions are satisfied: (1) the signal strength of each component's spectrum is proportional to that component's concentration and (2) the component spectra add linearly. Note that in some cases the signal does not satisfy these conditions but a function of the signal does. For example, in transmission-absorption measurements where the Lambert-Beer law applies the transmitted signal does not satisfy (1) or (2) but the logarithm of the transmitted signal (i.e., the absorption spectrum) does.
Important examples of samples that do not satisfy the linearity assumptions above are turbid or heterogeneous samples. In optical spectroscopy, radiation that propagates through a turbid sample is scattered and its path is altered. The alteration of the light path has two possible consequences; the light may miss the detector and not be detected at all or the detected, scattered light may have traversed the sample in a randomly circuitous way. Both consequences affect the absorption spectra, and therefore affect concentration estimates (particularly in applications that require fixed, known sample path lengths in order to unambiguously determine component concentrations). The latter consequence means that the detected signal is an average of measurements of the sample at multiple path lengths. Further, if the turbid sample is heterogeneous and has localized, rather than uniformly, distributed absorbing components some light rays may pass through undiminished (zero path length) and be neither scattered nor absorbed. This will also contribute to erroneous interpretation of the sample's absorption spectrum. A particular example of this arises in the analysis of hemoglobin content in blood and we will use this illustrative example throughout since it contains all the salient features that this technique addresses. However, it should be clear that the work described in this patent is not limited to this application.
Whole blood is the natural state of blood consisting of virtually transparent plasma within which floats red blood cells (that contain the absorbing pigment, hemoglobin, of interest), lipids, white blood cells, platelets, and a host of other objects. Several considerations must therefore be addressed in order to design an instrument that will provide an accurate measure of the hemoglobin content. One consideration is light scatter that traditionally has been minimized by breaking the red blood cells to form a homogenous mixture called lysed blood. While scatter is not completely eliminated with this approach (lipids, cell stroma, and other large particles are still present) the nonlinearities induced by the residual scatter are small enough to be neglected or treated with simple corrections to account for their affects. Other considerations are the sample cell dimensions and optical design to insure that the measurement takes place within an optimal absorption range (adequate signal-to-noise) and at fixed, known path length of the apparatus, respectively. Absorption can be adjusted by known dilution of the blood or by appropriate choice of sample cell dimensions (i.e. path length). The optical designer must insure that the incident source light is adequately collimated to create a unique path length and that enough transmitted light is collected to satisfy signal strength requirements.
With the desire to measure unaltered, whole blood, prior attempts to perform analysis on turbid, absorbing samples have required specialized apparatus that allow for the collection of the directly transmitted light and as much of the scattered light as possible. It would be desirable to perform accurate analysis of turbid, absorbing samples without having to precondition the sample or without having to utilize specialized light collection devices. Further, it would also be desirable to relax the stringent requirements on optical design (i.e., collimated probe light beam) in the analysis of non-turbid samples. The invention described in this patent provides for accurate analysis of samples without the above-mentioned constraints.